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Anal. Chem. 1984, 56, 1089-1096
(7) Hanaoka, Y.; Murayama, T.; Muramoto, S.; Matsuura, T.; Nanba, A. J . Cbrornatogr. 1982, 239, 537-548 (Figure 14).
(3) Tijssen, R. Sep. Sci. Techno/. 1978, 73, 681-722. (4) Reiin. J. M.: van der Linden, W. E.; Poppe, H. Anal. C h h . Acta 1981, 726, 1-13. (5) Pohl, C. A.; Johnson, E. L. J . Chrornatogr. Sci. 1980, 78, 442-452. (6) Stevens, T. S.; Davis, J. C.; Small, H. Anal. Chem. 1981, 53, 1488-1492. \
I
for review November
77
lgg3* Accepted February
2, 1984.
o-Phthalaldehyde Derivatives of Amines for High-speed Liquid Chromatography/Electrochemistry Laura A. Allison, Ginny S. Mayer, and Ronald E. Shoup* Bioanalytical Systems, Inc., Technical Center, West Lafayette, Indiana 47906
The conventlonal problems of derlvatlve stablllty and fluorescence quantum ylelds In the case of amlnes reacted with o-phthalaldehyde and varlous thlols were clrcumvented by a liquld chromatography/electrochemlstry (LCEC) approach. The degradative hydrolysls of the Ssubstltuted IsoIndole was Investigated by varylng the sterlc bulk of the thlol used In the o-phthalaldehyde reagent. No appreciable lmprovement In stability accrued until the bulky terf-butyl group was Incorporated, whereupon half-llves In excess of several hours were appreclated. I n contrast to fluorescence, the thlol had llttle Influence on the electrochemistry of the derlvatlve, whlch was characterired as a very rapld, Irreversible oxldatlon of the Isolndole. Gradient Separations of both the mercaptoethanol and fert -butyl derlvatlves on short 3-pm reversed-phase columns permitted LCEC detectlon llmlts of less than 500 fmol In the gradlent mode. Separations of 22 amlno aclds could be accompllshed In under 10 mln, but reequlllbratlon lengthened the total analysis time to about 30 mln. Detectlon llmlts were lowered to 30-150 fmol In the lsocratlc mode.
0-
The quantitation of amines in varied samples is a problem that has prompted the development of numerous analytical schemes. The most successful approaches have been those which couple derivatization of amines with a separation based on liquid chromatography, as illustrated by the amino acid method first proposed by Spackman, Stein, and Moore ( I ) . Their amino acid analyzer was basically a specialized liquid chromatograph, utilizing ion-exchange chromatography and derivatization. With the advent of high-performance liquid chromatography, newer systems have emerged which capitalize on the higher level of chromatographic capability. Generally, detection of chromophoric derivatives combined with continuous two-component gradients on hydrophobic octadecylsilyl or hydrophilic cation exchange stationary phases have been employed. For amino acids, derivatization schemes include those based on ninhydrin (Z), o-phthalaldehyde (3-6), fluorescamine (Z), dansyl chloride (7),dabsyl chloride (8),and 7-fluoro-4-nitro-2,1,3-benzoxadiazole (NBD-F) (9). In particular, fluorescence methods utilizing o-phthalaldehyde as a derivatizing reagent in both the pre- and postcolumn modes have become popular, although they continue to face certain limitations. The reaction of o-phthalaldehyde (OPA) with amino acids in the presence of a thiol reducing agent to produce fluorescent products was first re0003-2700/84/0356-1089$0 1.50/0
ported by Roth (10); the fluorescent products were subsequently determined (11)to be l-(alkylthio)-2-alkylisoindoles (I). The S-substituted isoindoles formed by the reaction of
amines with OPA and either p-mercaptoethanol or ethanethiol are amenable to reversed-phase liquid chromatography in conjunction with precolumn derivatization. However, a significant problem with precolumn OPA approaches has been the instability of the derivatives (5, 6, 12), causing careful timing or even instrumental automation to ensure reproducibility. Postcolumn derivatization schemes utilizing OPA must scrupulously avoid impurities in the reagents and mobile phase buffers which can contribute to high background fluorescence (13). Postcolumn schemes also result in some loss of both resolution and sensitivity due to mixing the mobile phase with diluent (14, 15). Several researchers have investigated the possibility of enhancing OPA derivative stability by varying the structure of the thiol compound (15, 16). Although a number of alternate thiols were utilized in the OPA reaction, some of which improved stability, it was found that the fluorescence properties of the derivatives were also markedly dependent on the structure. The trade-offs between stability and fluorescence prevented a true optimization of the OPA/thiol derivatization reaction. Recently, it was reported by Joseph and Davies (17) that OPA/P-mercaptoethanol derivatives of amino acids undergo anodic oxidation a t moderate potential, permitting the use of liquid chromatography/electrochemistry (LCEC) for their determination. The present study was suggested by our speculation that the electrochemical properties of the derivative will be far less susceptible than fluorescence to changes in the derivative's structure. Hence, alternative thiols were utilized to search for a more stable isoindole derivative which would be suitable to a simple, precolumn LCEC method without stability limitations. A second important advantage to the implementation of an electrochemically active derivatization is the compatibility of LCEC with high-speed chromatographic separations, such as those obtained with short, small particle size columns. Since detection occurs on a surface, rather than homogeneously in solution, it is convenient to reduce the cell volume with little degradation in detector performance. Concurrent with our study of alternate thiols, 0 1984 Amerlcan Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 56, NO. 7, JUNE 1984
the use of high-speed LCEC for derivative detection was therefore investigated. These amino acid derivatives were used as model systems. EXPERIMENTAL SECTION Apparatus. Isocratic liquid chromatographic experiments on OPA derivatives were performed on a Bioanalytical Systems LC-304. Gradient separations utilized a Spectra-Physics 8700 ternary solvent delivery system with a Bioanalytical Systems LC-4B detector and Hewlett-Packard 3390A integrator. All chromatography was performed with Perkin-Elmer HS-3 3 pm C-18 columns (100 X 4.6 mm); specific mobile phases and flow rates will be described in the text. A Bioanalytical Systems prototype detector cell was used which placed a glassy carbon working electrode, reference electrode, and auxiliary electrode in the vicinity of the thin-layer region. The potential of the working electrode was maintained at +0.70 V vs. Ag/AgCl, unless otherwise indicated. Thiol measurements were obtained with a Bioanalytical Systems, Inc., LC-154, modified to exclude oxygen from the system. All Teflon tubing was replaced by stainless steel. The mobile phase was heated to 50 "C for 30 min in a reflux apparatus and continuously bubbled with nitrogen to maintain an oxygen-free atmosphere (18). The reflux apparatus served as the mobile phase reservoir and remained under slight positive Nz pressure to exclude oxygen at all times. The working electrode was a BAS TL-6A Hg/Au amalgam electrode, prepared according to the manufacturer's instructions and maintained at +0.15 V vs. AgIAgC1. Electrochemical investigations employed either a Bioanalytical Systems, Inc., (3-27 voltammographor BAS-100 electrochemical analyzer. Acetonitrile was dried over alumina which had been previously heated to 300 "C overnight. Mass spectrometric data were obtained on a Finnigan 4000 mass spectrometer in either electron impact or chemical ionization modes. Fluorescence studies were performed with a Perkin-Elmer MPF-44A fluorimeter. Reagents. Borate Buffer. A total of 9.5 g of sodium tetraborate decahydrate was dissolved with heating in 250 mL of deionized, distilled water. The pH was adjusted to 9.5 with 1 M sodium hydroxide. o-Phthalaldehydel Thiol Buffered Reagents. A total 540 mg of o-phthalaldehyde was dissolved in 10 mL of methanol. The OPA solution and 400 pL of a given liquid thiol @-mercaptoethanol, ethanethiol, 3-mercaptopropionic acid, 2-mercaptopropionic acid, and 2-methyl-2-propanethiol(tert-butylthiol)) was diluted to 100 mL with pH 9.5 borate buffer. For the crystalline thiols, 870 mg of mercaptosuccinic acid was used per 100 mL of reagent, while 894 mg of thiosalicylicacid was used. An additional 10% methanol was utilized where necessary to aid solution of the thiol in the borate buffer. Buffered reagents were prepared in a hood and stored at room temperature, since refrigeration caused the undesirable precipitation of borate buffer salts. /3-Mercaptoethanol reagent was stable for up to 1 month, while tert-butylthiol reagent was restored by adding 200 pL of tert-butylthiol weekly. Here the designation RSH will refer to any of these alkylthiols. Mobile Phase. Mobile phases were prepared with deionized water (Nanopure, Barnstead), filtered through 0.2-pm Nylon 66 membranes (Rainin), and degassed prior to use. Gradient mobile phases were continually sparged with research grade helium to prevent outgassing. Standard Solutions. Amino acid standard solutions were prepared to a concentration of 1.0 mM in 50% methanol/50% water (containing 0.1% EDTA, pH 4-6) and refrigerated. Dilutions were prepared as necessary in methanollwater. Procedures. Derivatization with Protection of Cysteine. Twenty microliters of sample (standard solution or prepared sample) was placed in a small conical reaction vessel, to which was added 20 p L of 80 mM methyl iodide solution in methanollwater (5050, v:v) and 10pL of 3 M aqueous sodium hydroxide. After 10 min, 10 pL of 3 M aqueous perchloric acid and 80 pL of OPA/RSH buffered reagent were mixed with the solution. Precisely 2 min after addition of buffered reagent, 1-2 pL of the derivatization mixture was injected onto the LCEC system. Deriuatization without Protection of Cysteine. Twenty microliters of sample was mixed with 100 pL of OPA/RSH buffered
Figure 1. Hydrodynamic voltammograms for o-phthalaldehydelbmercaptoethanol derivatives of y-aminobutyric acid (O),methionine (A),tryptophan (0),and leucine (A)in a mobile phase consisting of 77% 0.05 M phosphate buffer, pH 7.0, and 1 mM Na,EDTA with 23% acetonitrile. 4 is defined as the ratio of the peak current to the limiting current.
reagent. Injection of 1-2 pL was made at precisely 2 min after mixing, unless otherwise noted. Preparation of Beer and Brain Samples. Beer was sonicated for 20 min to remove carbon dioxide and then derivatized as above, using methyl iodide for protective alkylation of cysteine. Whole rat brains were obtained from male Wistar rats by decapitation; the brains were rapidly excised and immediately frozen over dry ice. The tissues were then homogenized in cold methanol/water (50:50, v:v), using a Polytron apparatus and ice bath. The soups were centrifuged at 16-2OOOOg for 20 min at 4 "C and the supernatant was collected, diluted to known volume, and then derivatized per above. Synthesis of 1-(tert-Butylthio)-2-n-propylisoindole for Electrochemical Study. The stable derivative was prepared by the method of Simons and Johnson (19,ZO); the uncorrected melting point was 57.4-58.5 "C (lit. 58.3-59.0 "C). Electron impact mass spectral analysis produced major peaks at 247 (M+),191 (M+isobutylene), 148 (191- C3H7),57 (tert-butyl), and 41 (57 - CH,). The chemical ionization spectra with methane as reagent gas revealed 248 (MH') and 192 (MH' - isobutylene) as the major peaks. Both were in agreement with previous reports (20). Residual tert-butylthiol was measured by LCEC with a Hg/Au working electrode at +0.15 V. Synthesis of Non-S-Substituted Isoindoles. 1,3,4,7-Tetramethyl-1H-isoindole and 1,2,3,4,7-pentamethylisoindolewere formed by refluxing hexane-2,5-dione with 2,5-dimethylpyrrole and 1,2,5-trimethylpyrrole in glacial acetic acid (21). Upon basification, the crude products were sublimed as needed for purification before voltammetric studies. These compounds were relatively unstable upon exposure to air; the crude products discolored significantly in less than 5 min. RESULTS AND DISCUSSION o -Phthalaldehyde/B-Mercaptoethanol (OPAIB-MCE)
Derivatives. Before attempting to modify the reagents for an improved derivative, the established o-phthalaldehydel /3-mercaptoethanol chemistry was evaluated for use with liquid chrornatographylelectrochemistry,specifically with high-speed separations. Eight amino acid derivatives including a-aminobutyric acid (AABA), y-aminobutyric acid (GABA), methionine, phenylalanine, leucine, isoleucine, tryptophan, and valine were selected for preliminary studies conducted with an isocratic LCEC system. The eight derivatives were chromatographically separated with a mobile phase of 23% acetonitrile/77% 0.05 M phosphate buffer, pH 7.0, with 1 mM NazEDTA. The hydrodynamic electrochemical behavior observed (Figure 1) demonstrated relatively low half-wave potentials (EljzN 0.45-0.60 V vs. AgIAgC1). The tryptophan derivative shows a lower half-wave potential than other de-
ANALYTICAL CHEMISTRY, VOL. 56, NO, 7, JUNE 1984
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Figure 2. LCEC response as a function of reaction time for 0 phthalaldehydel&mercaptoethanol derivatives of amino acids. Derivatives shown represent y-amlnobutyrk acid,).( a-amh?obutyricackl (e),valine (m), methionine (A),tryptophan (O), Isoleucine (0),phenylalanine (O), and leucine (A).
rivatives (El,: 0.33 V), presumably induced from the native electrochemical activity of free tryptophan. A working potential of +0.70 V was selected for subsequent experiments, where the background currents were generally only 1-3 nA. Figure 2 shows the results of a study to determine the optimum reaction time for the OPA/@-MCE derivatization. Separate reaction mixtures were incubated for predetermined times; the resulting derivative peak heights were plotted vs. reaction time. Derivatization was maximal in each case by 1 min, a reaction time of 2 min was selected to permit adequate time to prepare chromatographic injections. Minimum detectable quantities in the isocratic system ranged from 30 to 150 fmol of amino acid injected, based on a signal-to-noise ratio of 3. A high speed gradient separation was developed for the analysis of mixtures of 21 amino acids in approximately 10 min (Figure 3A). The solvent system consisted of a mixture of a buffer (0.05 M NaC10,/0.005 M trisodium citrate (pH 5.00)), methanol, and tetrahydrofuran (THF). Contrary to previous reports, the compatibility of LCEC with gradient elution is excellent. Gradient runs using standard amino acid OPAIB-MCE derivatives have shown that quantities as low as 500 fmol are detected. The amino acid derivatives elute from the C-18 column on the basis of the hydrophobicity of the amino acid side chains; at pH 5.0, the elution order is that depicted in Figure 3A. Coelution of GLY and THR occurred when methanol was the only organic modifier, but resolution was obtained with the inclusion of a small percentage of THF in the mobile phase. It was possible to more widely disperse the OPAIP-MCE derivatives over the 10-min elution time, by increasing the organic content (>20%) of the mobile phase and using a more gradual gradient profile. However, the peak shapes of the early eluting derivatives, particularly ASP, were broadened and tailed under these conditions, without a significant increase in the resolution between closely eluting pairs such as glutamic acid and glutamine or glycine and threonine. The tailing is presumably caused by injection of the pH 9.5 borate reaction mixture into the weakly buffered pH 5.0 mobile phase; the aspartic and glutamic acid derivatives are momentarily placed in a nonhomogeneous mobile phase and begin to elute as two pairs of peaks. Reducing the initial organic content to 20% reduces migration of the derivative in either form until pH equilibrium is reestablished; then the
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gradient program takes over the separation to deliver a reproducible elution pattern. An alternate gradient program used a pH 5.8 buffer and significantly more T H F as the controlling elution factor (Figure 3B). The higher starting pH moved ASP and GLU to the front of the separation and inverted the positions of GABA and methyl-CYS, and HIS and GLN. Arginine moved in relative to THR and ALA. A pH 5.5 buffer was also attempted but GLU coeluted with ASN. The separation of the quartet MET/VAL/TRP/PHE was highly dependent upon the nature of the solvent program. Use of only methanol gradients caused the elution order to be MET/TRP/VAL/ PHE, with excellent resolution. Using only T H F inverted T R P and VAL. By starting at 10% methanol in A, we maintained the separation and the order was MET/VAL/ TRP/PHE. Using 20% methanol in A was sufficient to cause TRP/VAL to coelute. A high sensitivity blank run under the conditions of Figure 3B is shown in Figure 3C. The tailing peak is due to the presence of excess B-MCE in the reagent; no other appreciable peaks are evident. The rise in background current over this gradient program amounted to less than 2-5 nA at 700 mV, over several different batches of mobile phase. Reproducibility of the separation was found to be a function of two major factors. First, a period of reequilibration for the column with the initial mobile phase was required before subsequent injections. The best results were obtained when injections were made consistently at 30-min intervals. We suspect that the appreciable capacity factor of THF under the initial conditions may be casual. A second important consideration involved the limited capability of this particular gradient pump to adequately deliver the rapidly changing solvents required for the high-speed separation. With a relatively large 0.19 in. diameter piston, the volume delivered per stroke is too large to provide adequate temporal resolution of the composition, causing quantitization error. This disadvantage is stridly caused by a hardware limitation and could be eliminated by suitable instrumental design (e.g., using 2 mm diameter pistons). With these limitations in mind, we evaluated the precision and linearity of the system. For the 19 amino acids evaluated, the coefficients of variation for replicate reaction mixtures (n = 6) ranged from 2.8 to 9.2% for peak area and from 0.52 to 0.75% for retention time. The linearity was evaluated by using mixtures containing 1.67-167 pmol of each amino acid per injection. All compounds yielded correlation coefficients ranging from 0.993 to 0,9999. Slopes varied according to intrinsic chemical response factors, detector gain, and gradient elution times (range: 187 to 829 area counts/pmol injected). Because of its thiol functionality, cysteine reacts with OPA as both a thiol and amine, and a single LCEC detectable derivative is not obtained. Cysteine, however, will react conventionally with OPA/thiol reagents when the thiol group is protected. We selected alkylation of cysteine with methyl iodide as a protection mechanism, because the OPAIP-MCE derivative of S-methylcysteineeluted in an uncongested region of the gradient chromatogram (Figure 3A), as predicted by interpolation of the hydrophobic behavior of alanine and methionine. The methyl iodide was added to the sample in conjunction with a sodium hydroxide solution, which raised the pH to deprotonate the thiol for an S N 2 attack on methyl iodide. It was necessary to add equimolar perchloric acid prior to derivatization with OPA/RSH buffered reagent, as the excessively low pH caused major distortion in chromatographic peak shapes when this step was omitted. The OPA/p-MCE derivatization chemistry was applied to the determination of amino acid levels in commercially available beer samples. The amino acid content of malted
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Flgure 3. (A) High-speed chromatographic separation of OPAIP-MCE derivatives of 21 amino acids, 167 pmol of each amino acid derivative Injected. Gradient conditions were as follows: (A) 20% MeOH, 80% 0.05 M NaCi0,/0.005 M sodium citrate (pH 5.0); ( 6 )76% MeOH, 19% buffer, 5% THF; flow rate 1.5 mL/min. Program: 0 min, 100% A/O% B; 8.0mln, 15/85; 9.0 min, 0/100; 9.5 min, 0/100; 9.6 min, 10010. Stationary phase was Perkin-Elmer HS-3 3-pm C, column, 100 X 4.6 mm. (B) Chromatogram of 22 amino acids under alternate gradient, 113-258 pmol of each derivative injected. Gradient conditions were as follows: (A) 10% MeOH/9O% (0.05 M NaC10,/0.05 M sodium citrate (pH 5.80)); (B) 80% MeOH/20% buffer (pH 5.50), (C) 100% THF; flow rate 1.3 mL/min. Program: 0 min, 90% A/8% 812% C; 4 min, 70/28/2; 5 min, 55141.713.3; 7 min, 55/41.7/3.3; 13.5 min, 30/50/20; 15 min, 20/60/20; 18 min, 20/60/20. (C) Water "blank" was derivatized and chromatographed under conditions of Figure 3B, except at 10-fold higher detector gain.
Table I. Stability of OPA/p-MCE Derivatives % of Initial Response Remaining after 40 Minutes
0-25% OPA Amino Acids in Beer
glycine taurine GABA
ornithine lysine
150 nA 1
0
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6
a
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Flgure 4. Chromatogram of OPAIP-MCE derivatives of the amino acids in a commercial beer sample. Gradient conditions were those of Figure 3A.
barley is of primary importance to fermentation and yeast metabolism. Active yeast growth requires nitrogen uptake and this is drawn primarily from the amino acids. A rapid means of amino acid analysis is therefore necessary for un-
50-85%
85-100%
alanine serine leucine methionine arginine tryptophan histidine
phenylalanine aspartic acid valine glutamic acid glu tarnine isoleucine tyrosine
derstanding overall yeast performance in relation to the brewing process, the effect of barley variety on fermentation, and the development of new malting varieties. A typical chromatogram of the OPA/P-MCE amino acids in a beer sample under the conditions of Figure 3A is shown in Figure 4. Amino acid concentrations ranged from 33 pM (glutamine) to 1480 pM (alanine) in the sample, and were readily determined by using this methodology. Recoveries from beer were ascertained by spiking the beer samples with 0-15 nmol of each amino acid. The slope of the standard addition curve (four points) was compared to that obtained for spiked water samples processed under the same conditions. The slopes would be identical if 100% recovery were the case. Over 23 amino acids investigated, the average recovery was 100.4 i 1.8%,and the range of recovery varied
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Table 11. Effect of Thiol Structure on Stability % difference in responsea
30 min thiol HSCH,CH,OH HSCH~CH;
60 min
GABA
MET
GABA
-95 -100
t 5.3
-96
MET
comments
-23
multiple peaks CH(COOHMSHEH,COOH t 3.4 -100 t 8.2 -86 HsCH,CH~’COOH multiple peaks HSCH,CH,COOCH, t 1.0 -70 t 0.5 -50 2-mercaptobenzoic acid multiple peaks CH,CH(SH)COOH -6.3 -0.7 -10 0 (CH,),CSH a Defined as the percent change in peak height for injections made at the designated time relative to those made 2 min after mixing reagent and sample. from 97.2% to 103.9% for individual compounds. The stability of OPA/P-MCE derivatives of amino acids was investigated by preparing a derivatization mixture and periodically monitoring the LCEC response for each amino acid (Table I). Stability of the derivatives was a significant problem for glycine, taurine, GABA, ornithine, and lysine, all of which dropped to less than 25% of their 2-min levels by 40 min. GABA and glycine were particularly notorious, with half-lives on the order of approximately 5 min. Continual sparging of derivatization mixtures with nitrogen did not enhance stability of the derivatives. An oxidative decomposition pathway is therefore unlikely. The stability problems observed are consistent with literature reports using fluorescence detection of OPA/@-MCEderivatives (5,6,12). The extremely rapid loss of derivative response suggests that a fair amount of degradation is taking place, even during the brief time period when the derivatives are traversing the analytical column. This has been previously suggested by Kucera (15). With this chemistry the only available approach to handling the lack of stability is the use of precisely timed derivatizations to minimize inter-sample variations. o -Phthalaldehyde Derivatives with Alternate Thiols. A proposed mechanism of breakdown of OPA derivatives occurs through hydrolysis of the isoindole at the 1-position (16).In light of this evidence and our observation that several of the least stable OPAIP-MCE amino acid derivatives are those with relatively short, linear side chains, we suspected that steric bulk of the amino acid side chain may be a factor in the stability. One way to provide this attribute for all amino acid derivatives is to use a bulky thiol. Substitution on an almost arbitrary basis is possible, since there is no electrochemical reason for anticipating a decreased oxidative signal from the isoindole group due to different aliphatic side chains. Derivatives of y-aminobutyric acid and methionine were further investigated as model adducts possessing low and moderate stability, respectively. A broad spectrum of thiols were considered as substitutes for 0-mercaptoethanol in the OPA/RSH derivatization approach. Table I1 outlines the results for eight thiol compounds, including P-mercaptoethanol for reference. Several cases produced anomalous, unusable results which merit comment. Derivatization mixtures utilizing mercaptosuccinic acid gave multiple, closely eluting peaks for the two amino acids, while 2-mercaptopropionic acid showed two apparent derivative peaks for each amino acid. Methyl 3mercaptopropionate produced six peaks from a mixture of GABA and methionine, suspected to represent two derivative peaks for each amino acid (one rapidly degrading and one relatively stable) plus one “intermediate” compound for each amino acid that grew to a maximum over time and then decreased. It was also suspected that these commercially purchased thiols were contaminated with additional thiols which
E (Volts vs. AgIAgCI)
- 0.2 - 0.4
- 0.6
0
- 0.8
-
1.0
-
Flgure 5. Hydrodynamic voltammograms for 0-phthaiaidehydelterf butylthiol derivatives of y-aminobutyric acid (O), methionine (A), tryptophan (0),and leucine (A)in a mobile phase consisting of 60% 0.05 M sodium perchlorate/0.005 M trisodium citrate buffer, pH 5.0 with 40% acetonitrile. 4 is defined as in Figure 1.
would account for the multiple peak observation. Purity was checked for each of the three thiols through a separate LCEC chromatographic system which was designed for thiol determination. All three compounds were found to contain no detectable traces (
